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Langmuir 2007, 23, 10816-10822
Oxidation of L-Cysteine at a Fluorosurfactant-Modified Gold Electrode: Lower Overpotential and Higher Selectivity Zuofeng Chen, Huzhi Zheng, Chao Lu, and Yanbing Zu* Department of Chemistry, The UniVersity of Hong Kong, Pokfulam Road, Hong Kong, China ReceiVed June 6, 2007. In Final Form: July 22, 2007 We describe the oxidation of L-cysteine (CySH) at a fluorosurfactant (i.e., Zonyl FSO)-modified gold electrode (FSO-Au). Significantly reduced anodic overpotential of CySH was observed. The FSO layer inhibited the adsorption of CySH and its oxidation products at the gold electrode surface, and the low coverage of the adsorbed thiol-containing species might account for the more facile electron-transfer kinetics of free CySH at low potentials. An electrochemical impedance spectroscopy study revealed the lower charge-transfer resistance of CySH oxidation at the FSO-Au electrode as compared to that at a bare gold electrode. Interestingly, although the FSO layer facilitated CySH oxidation, the anodic responses of other electroactive biological species such as glucose, uric acid, and ascorbic acid were generally suppressed. Furthermore, the modified electrode was capable of differentiating CySH from other lowmolecular-mass biothiols such as homocysteine and glutathione. The unique features of the FSO-Au electrode allowed for the development of a highly selective method of detecting CySH in complex biological matrices. The direct determination of free reduced and total CySH in human urine samples has been successfully carried out without the assistance of any separation techniques.
Introduction L-Cysteine (CySH), a sulfur-containing amino acid, plays an important role in living systems, and its deficiency is associated with a number of clinical situations, such as liver damage, skin lesions, slowed growth, and AIDS.1-3 The determination of CySH has been the focus of numerous research efforts.2-16 Compared to the fluorescent and UV/vis spectroscopic detection methodologies, the electrochemical technique has the inherent advantages
(1) Dro¨ge, W.; Eck, H. P.; Mihm, S. Immunol. Today 1992, 13, 211. (2) Shahrokhian, S. Anal. Chem. 2001, 73, 5972. (3) Wang, W.-H.; Rusin, O.; Xu, X. Y.; Kim, K. K.; Escobedo, J. O.; Fakayode, S. O.; Fletcher, K. A.; Lowry, M.; Schowalter, C. M.; Lawrence, C. M.; Fronczek, F. R.; Warner, I. M.; Strongin, R. M. J. Am. Chem. Soc. 2005, 127, 15949. (4) Spa˜taru, N.; Sarada, B. V.; Popa, E.; Tryk, D. A.; Fujishima, A. Anal. Chem. 2001, 73, 514. (5) Zen, J. M.; Senthil Kumar, A.; Chen, J. C. Anal. Chem. 2001, 73, 1169. (6) Nekrassova, O.; Lawrence, N. S.; Compton, R. G. Talanta 2003, 60, 1085. (7) Nekrassova, O.; Lawrence, N. S.; Compton, R. G. Electroanalysis 2004, 16, 1285. (8) Lock, J.; Davis, J. Trends Anal. Chem. 2002, 21, 807. (9) White, P. C.; Lawrence, N. S.; Davis, J.; Compton, R. G. Electroanalysis 2002, 14, 89. (10) (a) Andersson, A.; Isaksson, A.; Brattstro¨m, L.; Hultberg, B. Clin. Chem. 1993, 39, 1590. (b) Ivanov, A. R.; Nazimov, I. V.; Baratova, L. A. J. Chromatogr., A 2000, 870, 433. (c) Causse´, E.; Issac, C.; Malatray, P.; Bayle, C.; Valdiguie´, P.; Salvayre, R.; Couderc, F. J. Chromatogr., A 2000, 895, 173. (d) Huang, X.; Kok, W. T. J. Chromatogr., A 1995, 716, 347. (11) (a) Reynaud, J. A.; Malfoy, B.; Canesson, P. J. Electroanal. Chem. 1980, 114, 195. (b) Johll, M. E.; Williams, D. G.; Johnson, D. C. Electroanalysis 1997, 9, 1397. (12) (a) Vandeberg, P. J.; Johnson, D. C. Anal. Chem. 1993, 65, 2713. (b) Johll, M. E.; Williams, D. G.; Johnson, D. C. Electroanalysis 1997, 9, 1397. (c) Vandeberg, P. J.; Johnson, D. C. Anal. Chim. Acta 1994, 290, 317. (d) LaCourse, W. R.; Owens, G. S. Anal. Chim. Acta 1995, 307, 301. (e) Owens, G. S.; LaCourse, W. R. J. Chromatogr., B 1997, 695, 15. (13) Lawrence, N. S.; Davis, J.; Compton, R. G. Talanta 2001, 53, 1089. (14) (a) Salimi, A.; Hallaj, R.; Amini, M. K. Anal. Chim. Acta 2005, 534, 335. (b) Huang, X.; Kok, W. T. Anal. Chim. Acta 1993, 273, 245. (c) Park, J.; Shaw, B. R. J. Electrochem. Soc. 1994, 141, 323. (d) Qi, X. H.; Baldwin, R. P. J. Electrochem. Soc. 1996, 143, 1283. (e) O’Shea, T. J.; Lunte, S. M. Anal. Chem. 1994, 66, 307. (15) (a) Shi, G.; Lu, J.; Xu, F.; Sun, W.; Jin, L.; Yamamoto, K.; Tao, S.; Jin, J. Anal. Chim. Acta 1999, 391, 307. (b) Zhang, S.; Sun, W.; Zhang, W.; Qi, W.; Jin, L.; Yamamoto, K.; Tao, S.; Jin, J. Anal. Chim. Acta 1999, 386, 21. (16) (a) Zhao, Y. D.; Zhang, W. D.; Cheng, H.; Luo, Q. M. Sens. Actuators, B 2003, 92, 279. (b) Xu, J.; Wang, Y.; Xian, Y.; Jin, L.; Tanaka, K. Talanta 2003, 60, 1123. (c) Fei, S.; Chen, J.; Yao, S.; Deng, G.; He, D.; Kuang, Y. Anal. Biochem. 2005, 339, 29.
of simplicity, ease of miniaturization, high sensitivity, and relatively low cost.6,7 However, previous studies revealed a variety of challenges for the electrochemical detection of CySH. First, CySH oxidation at solid electrodes, such as noble metals and carbon, is usually plagued by sluggish electron-transfer kinetics; therefore, large overpotentials are required to attain reasonably good sensitivity.8,9 The high anodic potential applied may significantly reduce the detection selectivity, especially for biological samples. In addition, the coexistence of other lowmolecular-mass biothiols such as homocysteine (Hcy) and glutathione (GSH) may pose a further obstacle to the specific determination of CySH.9 A straightforward means of solving these problems is to employ separation techniques, such as HPLC and capillary electrophoresis, in conjunction with electrochemical detection.10 However, this greatly increases the test complexity and cost. Another difficulty in oxidizing CySH at noble metal electrodes results from the passivation of the electrodes by surface oxides formed at high potentials.9,11,12 Pulse electrochemical detection procedures have been developed to overcome this problem by cleaning the electrode in situ.12 Alternatively, a variety of chemically modified electrodes (CMEs) have been investigated in order to enhance the analytical signals. The modification layer on the electrode surface generally involves electron-transfer mediators, and thiol oxidation can be facilitated through electrocatalytic conversion. The studied mediators include electroactive organic species (such as benzoquinone, catechol, and N,N-diethyl-p-phenylenediamine)6,7,13 and inorganic species (such as ruthenium and cobalt complexes).14 A problem in such systems is the leaching of the redox mediators from the electrode surface, which may be alleviated by the addition of polymeric coatings.15 Some newly developed electrode materials, such as boron-doped diamond4 and carbon nanotubes,16 have also been employed for the oxidation of CySH. In spite of intensive study, the direct electrochemical determination of CySH in real biosamples has been less-often reported, mainly because of substantive interference. To achieve this attractive goal, the detection selectivity has to be greatly improved. On the one hand, the measurement must be performed at a
10.1021/la701667p CCC: $37.00 © 2007 American Chemical Society Published on Web 09/07/2007
Oxidation of L-Cysteine at a Modified Au Electrode
relatively low potential to avoid the responses of other electroactive species. On the other hand, what is more difficult is a means of differentiating CySH from other small biothiols, but this should be developed. Recently, we have been interested in applications of fluorosurfactant-modified gold and platinum electrodes in electroanalysis17 and have found a number of unique features of the modified electrodes. Generally, electron-transfer processes would be suppressed by the adsorbed fluorosurfactant molecules whereas some electrochemical reactions (e.g., the oxidation of tertiary amines) could be greatly enhanced.17a Fluorosurfactants are well known for their high surface activity as well as chemical and thermal stability in harsh environments. Their adsorption behaviors at the electrode/electrolyte interfaces have been reported.18-23 As compared with their hydrocarbon counterparts, the adsorption of fluorosurfactants is weaker at a hydrophobic electrode (e.g., Hg) but stronger at a hydrophilic electrode (e.g., Pt and Au). In the present report, the electrochemical oxidation of CySH at a gold electrode modified with nonionic fluorosurfactant species Zonyl FSO was studied. A significant negative shift of the CySH oxidation potential has been observed, which allowed for the amperometric detection of CySH at ∼0.25 V versus SCE in neutral solution. Signals of diverse electroactive species that could interfere with CySH detection were greatly suppressed. Furthermore, the oxidation wave of CySH could be clearly separated from those of other small biothiols. The lower overpotential and higher selectivity of CySH oxidation at the FSO-modified electrode provide a new means to determine CySH directly in biological matrices. Experimental Section Chemicals. Zonyl FSO-100 (F(CF2CF2)1-7CH2CH2O(CH2CH2O)0-15H), Triton X-100 (4-(C8H17)C6H4(OCH2CH2)nOH), 20 standard
amino acids, L-cystine, DL-homocysteine, and glutathione were purchased from Aldrich. Other chemicals were analytical reagent grade and were used as received. All solutions were prepared with deionized water (Milli-Q, Millipore). The pH of the phosphate buffer solution (PBS) was adjusted with concentrated NaOH or phosphoric acid. Apparatus. All of the electrochemical measurements were performed on a CHI 760B electrochemical workstation (Chenhua Instruments, Shanghai). The three-electrode system consisted of a working electrode, a saturated calomel reference electrode (SCE), and a coiled Pt wire counter electrode. Procedures. Prior to the experiments, a bare Au electrode of 2 mm diameter was wet polished with 0.05 µm Al2O3 powder to obtain a mirror surface, followed by sonication in distilled water for 10 s, and was subjected to repeated scanning in a wide potential range in 0.1 M H2SO4 solution until reproducible voltammograms were obtained. The FSO-modified Au electrode (FSO-Au) was prepared by dipping the pretreated Au electrode into a 5 wt % FSO aqueous (17) (a) Li, F.; Zu, Y. Anal. Chem. 2004, 76, 1768. (b) Zu, Y.; Li, F. Anal. Chim. Acta 2005, 550, 47. (c) Zheng, H.; Zu, Y. J. Phys. Chem. B 2005, 109, 12049. (d) Zheng, H.; Zu, Y. J. Phys. Chem. B 2005, 109, 16047. (e) Chen, Z.; Zu, Y. J. Electroanal. Chem. 2007, 603, 281. (18) Juhel, G.; Beden, B.; Lamy, C.; Leger, J. M. Electrochim. Acta 1990, 35, 479. (19) (a) Cachet, C.; Chami, Z.; Wiart, R. Electrochim. Acta 1987, 32, 465. (b) Cachet, C.; Saidani, B.; Wiart, R. J. Electrochem. Soc. 1991, 138, 678. (c) Cachet, C.; Keddam, M.; Mariotte, V.; Wiart, R. Electrochim. Acta 1992, 37, 2377. (d) Cachet, C.; Keddam, M.; Mariotte, V.; Wiart, R. Electrochim. Acta 1993, 38, 2203. (e) Cachet, C.; Keddam, M.; Mariotte, V.; Wiart, R. Electrochim. Acta 1994, 39, 2743. (f) Cachet, C.; Wiart, R. Electrochim. Acta 1999, 44, 4743. (20) (a) Tsuchiya, S.; Inoue, T. Japanese Patent 02,236,967 (90,236,967); Chem. Abstr. Jpn. 1993, 114, 85389d. (b) Myake, S. Japanese Patent 05,121,089 (93,021,089); Chem. Abstr. Jpn. 1993, 119, 99935u. (21) Shi, Z.; Zhou, Y. H.; Cha, C. S. J. Power Sources 1998, 70, 205. (22) Cha, C. S.; Zu, Y. Russ. J. Electrochem. 1995, 31, 796. (23) Cha, C. S.; Zu, Y. Langmuir 1998, 14, 6280.
Langmuir, Vol. 23, No. 21, 2007 10817
Figure 1. Cyclic voltammograms of FSO-Au (-) and bare Au (---) electrodes in 0.15 M PBS (pH 7.0). Scan rate, 10 mV/s. (Inset) Cyclic voltammograms obtained in a narrow potential range. solution for 2 min, followed by a thorough rinsing with distilled water. A Triton-modified Au electrode (Triton-Au) was obtained by a similar procedure. CySH solutions were prepared with deaerated water immediately before use. Cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were performed with a scan rate of 10 mV/s. Electrochemical impedance spectroscopy (EIS) was conducted in the frequency range from 0.01 Hz to 100 kHz with a voltage perturbation amplitude of 5 mV. The data obtained were analyzed using the fitting program in Zview software (Solartron Metrology Ltd.). Amperometric measurements were carried out by applying a fixed potential of 0.25 V to stirred solutions. Unless stated otherwise, 0.15 M PBS (pH 7.0) was used for all electrochemical measurements. Human urine samples were collected in the morning from healthy adult males. Free reduced CySH (fCySH) was analyzed immediately after sample collection. For the determination of total CySH (tCySH, including protein-bound, free oxidized, and free reduced CySH), the disulfide bonds were reduced by adding 600 µL of a freshly prepared 1.5 M NaBH4 solution (containing 0.05 M NaOH) and 60 µL of n-octanol to 2400 µL of urine. After being mixed, the solution was incubated in a 40 °C water bath for 30 min under gentle stirring. The reduced mixture was then centrifuged at 10 000 rpm for 20 min. To the supernatant, an aliquot of 3 M HCl was added in order to decompose excess NaBH4 and adjust the solution pH to ∼7.0.24-26 The urine samples were diluted by a factor of 2 before the measurements.
Results and Discussion Voltammetric Study. Figure 1 shows the CVs of bare Au and FSO-Au electrodes in pH 7.0 PBS. In the potential region that is less positive than ∼0.8 V, no evident anodic current was observed at the FSO-Au electrode, indicating an inhibiting effect of the adsorbed FSO on electrode surface oxidation. A relatively large anodic peak appeared at ∼0.95 V, which could mainly result from the desorption of the FSO layer and the growth of the electrode surface oxides.17a As shown in the inset diagram of Figure 1, the double-layer charging current and the residue redox currents in the potential region between -0.05 and 0.5 V were obviously reduced by the adsorption of the FSO species. In the presence of 100 µM CySH, a clear oxidation wave with a peak/plateau potential (Ep) of ∼0.25 V was obtained at the FSO-Au electrode, as shown in Figure 2a. On the reverse sweep, no reduction wave appeared, consistent with the well-known (24) Chwatko, G.; Bald, E. J. Chromatogr., A 2002, 949, 141. (25) Melnyk, S.; Pogribna, M.; Pogribny, I.; Hine, R. J.; James, S. J. J. Nutr. Biochem. 1999, 10, 490. (26) Fiskerstrand, T.; Refsum, H.; Kvalheim, G.; Ueland, P. M. Clin. Chem. 1993, 39, 263.
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Figure 2. (a) Cyclic voltammograms of 100 µM CySH at FSO-Au (-), Triton-Au (---), and bare Au (‚‚‚) electrodes. (b) Cysteine oxidation current at 0.25 V (background signal subtracted) vs pretreatment time of the Au electrode in a 0.05 wt % FSO solution. Scan rate, 10 mV/s. Solution, 0.15 M PBS (pH 7.0).
electro-oxidation.27
irreversible nature of CySH Figure 2a also shows the CV of CySH at a bare Au electrode, where the anodic response was sluggish at low potentials (
